Abstract 14042: Characterization of Multi-Cell-Type Engineered Heart Tissues Containing Human iPSC-Derived Cardiomyocytes, Endothelial Cells, Fibroblasts, Smooth Muscle Cells and Macrophages

Abstract

Introduction: Induction of a pathological type of hypertrophy in human engineered heart tissues (EHTs) has not yet been fully achieved. We hypothesized that this limitation is caused by the unicellularity of EHTs, which contain only cardiomyocytes (CMs) and lack the interactions with non-myocytes. Thus, we aimed to generate EHTs implementing five native cardiac cell types, i.e., CMs, endothelial cells (ECs), fibroblasts (Fbs), smooth muscle cells (SMCs), and macrophages (Mϕ). Methods and Results: CMs were differentiated from hiPSCs by mesodermal induction followed by inhibition of Wnt-signaling. Mesoderm progenitors were also used to differentiate ECs via VEGF stimulation. Differentiation into SMCs and Fbs required Wnt pathway activation followed by stimulation with TGFβ+bFGF or bFGF only, respectively. Finally, Mϕ differentiation required hematopoietic/myeloid specification by IL-3 and M-CSF treatment. Successful differentiation was analyzed by electron microscopy (EM) and expression profiling of hallmark genes and proteins. For instance, Mϕs exhibited high expression of myeloid markers (98.0% CD68 + , 90.9% CD14 + ) whereas Fbs showed upregulation in genes such as POSTN, DDR2, COL1A1 (all >100 fold higher than iPSCs). Functionality of all cell types was demonstrated by multiple assays. For example, the native Mϕ function was demonstrated via degradation and phagocytosis assays while ECs exhibited fast and extensive tube formation in matrigel assays. Additionally, Fbs produced 4.6x more collagen after TGFβ stimulation than unstimulated controls. In effect, multi-cell-type EHTs were generated from 60% CMs, 20% ECs, 10% Mϕ, 5% SMCs and 5% Fbs. These EHTs were characterized by confocal microscopy, serial block-face scanning EM, gene expression profiling, and automated contractility measurements. Pilot experiments revealed that the addition of different cell types accelerated the development of EHTs which was associated with earlier onset of beating and shorter diameter of the EHTs. Conclusion: Successful application of cell differentiation protocols resulted in the integration of five cell types in a multi-cell-type EHT system. The full functional profile and reaction to a stress stimulus will be investigated in future experiments.